DESCRIPTION The trafficking of lysosomal hydrolases to lysosomes depends on the modification of Asn-liked oligosaccharides to permit binding to mannose 6-phosphate (M6P) receptors. The critical initial step in the specific addition of M6P to lysosomal hydrolases is catalyzed by the enzyme, UDP-N-acetylglucosamine: lysosomal enzyme N-Acetylglucosamine-1-phospho-transferase. Using a novel monoclonal antibody, bovine GlcNAc phospho-transferase (PTase) has been purified to homogeneity as a complex of three polypeptides of 166 kDa, 56 kDa and 51 kDa. The GlcNAc-PTase subunits are encoded by at least two cDNAs, one of which has been isolated. The remaining cDNA(s) will be isolated and characterized. Confirmation that these cDNAs represent the structural genes for GlcNAc-PTase will be obtained by immunoprecipitation with antipeptide antibodies or expression of epitope-tagged subunits and immunoprecipitation with anti-epitope antibodies. Subunit specific monoclonal and polyclonal antibodies will be generated using immunopurified epitope-tagged subunits as immunogen. Structure function studies will be initiated by determining the molecular basis for human mucolipidosis II (MLII) and ML III by RT-PCR, cloning and sequencing of GlcNAc-PTase subunit cDNAs from a panel of 17 patient-derived fibroblast lines. The subunits or structures responsible for lysosomal enzyme binding, UDP-GlcNAc binding, and GlcNAc-1-phosphate transfer will then be identified by expression of naturally occurring and constructed mutant GlcNAc-PTase cDNAs in ML II fibroblasts, followed by specific function assay. Structures critical for GlcNAc-PTase functions will be further defined by determining the epitopes identified by inhibitory monoclonal antibodies, and by construction and expression of site-directed mutant cDNAs. These studies will improve understanding of the mechanisms involved in lysosomal enzyme trafficking and provide essential reagents for the development of M6P targeted enzyme replacement therapies for lysosomal storage diseases.